Members of the voltage-gated superfamily of ion channels generate the action potential and govern neural excitability, muscle contraction, neurotransmitter release and visual and olfactory signal transduction. These channels include voltage-gated K+, Na+, and Ca++ channels, as well as channels gated by cytoplasmic ligands such as Ca++ and cyclic nucleotides. As a group these channels are capable of high flux rate, but range in ion selectivity, though they all exclude anions. The channels appear to have a similar overall structure and may well gate by similar means. Understanding the operational principles of any one of the members should provide important insights into how they all function. This understanding should help in the development of new therapies which target specific channels in the superfamily for treatment of diseases as various as cardiovascular disease, intractable pain, and myotonia. This proposal sets out to define how voltage-gated ion channels respond to changes in membrane potential by opening and closing their molecular gates. The gates control access of permeant ions to the pore (and thus transmembrane ion flux) at several different locations along the length of the pore. The precise locations, structures, and rearrangements of the voltage sensors and gates of these channels are, at best, only partly understood. Also not well understood are the mechanisms by which voltage sensors in each of a channel's four subunits act to open or close the channel gates. The goal of this proposal is to further our understanding of these issues and to obtain a structural map of the Shaker K+ channel in various functional states. The long- term goal is to reconstruct in four dimensions the protein motions that underlie voltage sensing, that open and close the channel gates, and that couple the voltage-sensing apparatus to the gates.